RP HPLC Purification of Small Molecules

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1 RP HPLC Purification of Small MoleculesLou ChengGood Morning Everyone!!!First I like to thank Paul, Kerri, Marvin, and everybody else to give me a chance to present here. Today, I like to talk about “RP HPLC purification of small molecules”. This work was part of my work at Astrazeneca or AZ in the past year, and it was the part I have permission to present out of AZ.Astrazeneca R&D Boston

2 Outline Reversed-Phase HPLC – Isocratic and GradientHPLC Basics: Classification, k, α, N, RsReversed-Phase HPLC – Isocratic and GradientAnalytical Method Development – Screening & OptimizationAnalytical Scale-up of Optimized HPLC MethodFrom Analytical Scale-up to PrepLC PurificationPrepLC Purification – Fraction Collection & RecoverySummaryThis is also a chance for me to share you my understanding and experience on this topic. I would appreciate any questions from you at any time during this presentation.First I like spend one minute or two talking about some HPLC basics, so that you can better understand my presentation. After that I will use some real Astrazeneca examples to explain principles of both isocratic and gradient RP HPLC. Then I will show you how I apply these basics and principles in the screening and optimization of the analytical HPLC method, as well as in analytical scale-up. After that I will discuss how to go from analytical to preparative and the fraction collection and recovery in prepLC separations.

3 HPLC Basics - ClassificationIncreasing polarityWater-insolubleWater-solubleNonpolarIonicNonionic polarMolecular Weight102103104Liquid Chromatograph can be classified into four categories according to the separation mechanism: Adsorption, Partition, ion exchange, and size exclusion. This classification does not include chiral and affinity chromatography. Normally, molecules with less than 1000 Dalton molecular weigh are considered as small molecules.105106

4 HPLC Basics - ClassificationIncreasing polarityWater-insolubleWater-solubleNonpolarIonicNonionic polarMolecular Weight102103104My experience focused on the Gel Permeation Chromatography for macromolecules and adsorption/partition for small molecules. Reversed-Phase High-performance Liquid Chromatography, or RP HPLC is the topic of this talk.105106

5 HPLC Basics – k, α, N, Rs tR1 tR2 t0 w1 w2 tRi - t0 k = t0 k2 α = k1Minutes10mVoltsThe retention factor or capacity factor, describes how long the molecule was retained on a column. Theoretically, it is the ratio of the number of molecules in the mobile phase to the number of molecules in the stationary phase. Optimum Retention Factor k for isocratic HPLC is 2 < k < 20 , corresponding to retention time between 1.2 min and 13.2 min for 5 cm HPLC columns.The selectivity or separation factor evaluates how well two peaks are separated. α larger than 1.1 is normally required for analytical purpose. For an analytical method developed for PrepLC separation, we desire selectivity as big as possible.Efficiency/Plate number is a measure of how narrow peaks are in relation to how long the compound is retained. Plate number is a leading indicator of how much the column could be overloaded. Plate number less than two thousand is usually unacceptable according to FDA guideline. For an analytical method developed for PrepLC separation, we desire plate number as big as possible.Resolution is a comprehensive measure of separation by considering all retention factor, selectivity, and column efficiency. The purpose of our method optimization is to maximize resolution for peaks of interest.Retention factortRi - t0k =t0k2α =k1SelectivityN = 162tWiEfficiencyRs = 0.25 (-1)N0.5kk + 1Resolution

6 Reversed-Phase HPLC – Isocraticlog k = log kw – SφEq. 1*k: retention factorkw: retention factor by 100% water as mobile phase (φ = 0)S: a constant for a given sample compoundφ : organic fraction (volume) in binary mobile phaseKw: HydrophobicityRP HPLC could be operated under isocratic or gradient conditions. In isocratic, the composition of the mobile phases remains constant during separation. The most often used mobile phase combinations are water-ACN or water-methanol. The retention in isocratic is defined by this equation over some practical range.Kw and S are sample’s HPLC properties. They can be used to predict retention under both isocratic and gradient conditions.As a chemist, I tend to relate the Kw to the hydrophobicity of the sample, and S to the retention sensitivity of the sample to the change of mobile phase. Higher the Kw, more hydrophobic the sample is. High the S, more sensitive the sample is to the change of mobile phase.S: Sensitivity to change of mobile phase strength*Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.

7 Isocratic RP HPLC - AZ Example 1Conditions: HPChem 10, XBridge C8, 4.6 × 50 mm,10 mM NH4Form/AcN, 1.0 mL /minRule of Adjusting Isocratic Retention by φ: 5%  Ξ 100 % kLog k = log kw – SφID:(BCL2)(MW = 426.5)(MW = 454.3)Code:(CoaD)Here are two real AZ examples. In this figure, triangles are for the logarithm of retention factor, and circles are for the retention factor. As you can see, both cases fit this linear relationship over the range studied. Judged from Kw and S, blue compound is more hydrophobic and more sensitive to the change of mobile phase than the red one. Besides, in both case, 5% increase in organic percentage would cause ~100% decrease in retention.

8 Isocratic RP HPLC - AZ Example 2log k = log kw – Sφ, 10 g (HE-TMK)Kw = 28S = 3.35Kw = 62, S = 3.67Kw = 72, S = 3.72Mw = 163Mw = 158+In the case of isomers, the Kw and S of the isomers are almost identical, as red and green samples indicated here. The starting material had a very different Kw and S and its prep separation by RP HPLC from its isomeric product was very easy. However, the prep separation of isomers was proven undoable, and their final separation was done by normal phase using pure silica column.Conditions: XBridge C18, 4.6 × 50 mm, 0.1% NH4OH/MeOHFlow Rate: 1.0 ml /min, room temperature.The Kw and S of the two isomers are too close to be separable by RP gradient runs.

9 Reversed-Phase HPLC – Gradientb = SVm/tGF log k0 = log kw - S0tR = (t0/b) log [2.3k0b(ts/t0) + 1] + ts + tDEq. 2*T0: column dead volumn; b: gradient steepness; k0: k at the beginning of the gradient;ts: value of tR for a nonretained solute; tD: dwell time for gradient elution;: change of organic percentage in the mobile phase; S: system constant;Vm: column dead volumn; tG: gradient time; F: flow rate.For Linear Solvent Strength (LSS) Gradient:*Ref: Practical HPLC Method Development, Lloyd Snyder, etc. Wiley, New York, 1997.*By assuming S = 4 for all small molecules, one gradient run is sufficient to resolve kwTwo gradient runs can solve kw and S, by assuming log k/φ linear relationshipAutomatic method developmentRetention Prediction (Drylabs, Chromsword)In gradient mode, the organic percentage changes normally linearly with time, the retention is given by this equation:The three important parameters in the equation are b, gradient steepness, k0, retention factor at the beginning of the gradient which is determined by the starting organic percentage in the mobile phase, and delta phi, the total change of organic percentage in the mobile phase during the run. These three can be used in gradient method optimization.By assuming S = 4 for all small molecules, one gradient run is sufficient to resolve kw; Two gradient runs can solve Kw and S. These two assumptions are protocols adopted by Drylabs or Chromsord software for automatic method development. Here I like to show you two real examples.

11 Reversed-Phase HPLC – Gradient Over IsocraticWhy Gradient?Flexibility (b, , 0) to optimize separation with minimal effects on efficiency (N)Samples with a wide k range, sometimes containing late-eluting interferences that can either kill the column or carryover to subsequent runsMore precise, robust, and automatableDilute solutions of sample dissolved in a weak solventWe routinely start our method development with gradient RP HPLC. Why? One reason I mentioned before is that we have more tools available to optimize separation in gradient that in isocratic. Besides, optimization by organic percentage in isocratic has more adverse effects on column efficiency.Samples we received normally have a wide k range, containing very hydrophilic and very hydrophobic components, and the later sometimes are late-eluting interferences that could kill the column or carryover to subsequent runs. No isocratic conditions can give acceptable k for all of these components. While under gradient mode, these components can elute under a full gradient, and thus provides protection for the columns and for the sequent runs.Also, isocratic retention is more sensitive to temperature, solvent mixing by the pump, and its precision, robustness, and automatability under prep conditions are not as good as gradient ones.An extrra advantage for gradient HPLC is that you can prepare your samples in a weaker solvent than the starting gradient such as 100% water.So, gradient RP run is always the best starting point for method development, even if the final method was isocratic or normal phased.Gradient RP run is the best starting point for method development

13 Criteria for Evaluating and Optimizing HPLC MethodsGeneral:Low k, low tailing factor, high NHigh α, high RsClient-specific:●●●●●●MPS/library (universal applicability)Fraction collection for one component, multiple components, or all components?Purity/Recovery?pH stability of the desired components?Amount of the sample (high loading)How to evaluate the screening results? How to optimize HPLC methods after screening?Generally, we look for low k, low tailing factor, and high N for peaks of interest. For critical band pairs, we look for high α, and high resolution.Besides, there are some client-specific criteria. If the method is developed for QC, resolution of all peaks with sufficient symmetry and efficiency is required. For reaction monitoring, baseline resolution of reactants and products is required. In this talk, the purpose of our method development is purification. So are we requested to collect one component or multiple components? Request of one component collection would cost less, compared to multiple component collection.What is the purity/recovery requested? The answer would determine how to collect fractions and how to combine fractions.Some clients may tell us their samples are unstable under acidic or basic conditions. In that case, we would avoid methods using these conditions.If the sample is from a library, then the method developed should have the potential to fit all library samples.If the amount of sample is more than 1.0 gram, we would focus on the method with the highest loading capacities.Some times these chemist-specific criteria keep changing. So, clear and timely communication with chemists is the prerequisite to a successful method development.Clear communication with clients is a prerequisite to successful method development

14 AZ Example of Screening Sequence (2252-026)Here is a real example of intelligent screening sequence for sample It is intelligent because the first standard injection is used as a control sample to check system suitability. The sequence would stop automatically if it fails system suitability test.

15 From Screening To PrepLC(QuinFF)0.8 mg/ml, XBridge C18 (4.6 × 50 mm)5-95% MeOH/10 mM HCOONH4, 5 minutes, 1.0 ml/min, 240 nm.Tracked by MSD*Rs1.82.2Optimization30-80% MeOHRs3.64.8Anal. Scale-up100 mg/ml, 4.6 × 100 mm XBridge C1830-80% MeOH, 10 min, 1 ml/minPrepLC19 × 100 mm XBridge C1830-80% MeOH, 10 min20 ml/min, 100 mg/mlBaseline ResolutionFor this sample, this method, 10 mM AmForm/MeOH with XBridge C18, stood out, because it gave the best resolution and separation factor for this critical band pairs. Besides, the product peak, as tracked by MSD detector, was symmetrical and has the highest plate number.The client wanted to collect the product peak only. So by fine-tuning the starting gradient, gradient steepness, and gradient range, we were able to maximize the resolution of these two critical band pairs from 1.8 to 3.6 and 2.2 to 4.8 respectively.We then further scaled up this separation on analytical HPLC and finally PrepLC with baseline resolution.

16 From Screening To PrepLCScale-up/loading100 mg/ml, 4.6 × 100 mm XBridge C1830-80% MeOH, 10 min, 1 ml/minScreening(QuinFF)0.8 mg/ml, XBridge C18 (4.6 × 50 mm)5-95% MeOH/10 mM HCOONH4, 5 minutes, 1.0 ml/min, 240 nm.Tracked by MSD*Rs1.82.2Optimization30-80% MeOH3.64.8PrepLC19 × 100 mm XBridge C1830-80% MeOH, 10 min20 ml/min, 100 mg/mlBaseline ResolutionFor this sample, this method, 10 mM AmForm/MeOH with XBridge C18, stood out, because it gave the best resolution and separation factor for this critical band pairs. Besides, the product peak, as tracked by MSD detector, was symmetrical and has the highest plate number.The client wanted to collect the product peak only. So by changing the starting gradient, gradient steepness, and gradient range, we were able to double the resolution of these two critical band pairs from 1.8 to 3.6 and 2.2 to 4.8 respectively.We then further scaled up this separation on analytical HPLC and finally purified the desired product on Gilson prep system with baseline separation.

17 Analytical Scale-up of Optimized HPLC MethodGoal: 1) Is the optimized analytical HPLC method good for preparative one?2) If it is, what is the maximum loading for touching-band separation?OptimizationENSynergi Hydro-RP (4.6 × 50 mm)30-60% MeOH/TFA, 5 min,1.0 ml/min, 240 nm, 0.6 mg/ml.Product (MW =471.5)Anal. Scale-upSynergi Hydro-RP (4.6 × 100 mm)30-60% MeOH/TFA, 10 min,1.0 ml/min, 240 nm, 12.5 ul, 160 mg/ml.ProductThe screening and optimization were always done under analytical concentrations of the sample, which is normally below 1.0 mg/ml. We want to know if the optimized method could be used under preparative conditions. This could be done through analytical scale-up experiments. In analytical scale-up, the sample is normally hundred times concentrated than analytical one. Because of this, we have to replace analytical flow cell by prep flow cell. Besides, we always try to use columns with the same length as the prepLC columns. These changes make analytical scale-up conditions very close to prep conditions.The analytical scale-ups can answer following two questions: 1) is the optimized analytical HPLC method good for preparative one? 2) if it is, what is the maximum loading for touching-band separation?Sometimes, the optimized analytical is good for the prep separation, as indicated in the previous slide. But very often, the optimized analytical is not that good for prep. Here comes an example. In this optimized method, the resolution from the best screened method were already maximized. However in its scale-up, the efficiency and resolution were not optimized, and the touching-band loading was only 12.5 ul. We have 5 grams in 30 ml solvents, and it means it would take 125 injections for the prep separation. Clearly, this method is not practical for 5g separation.This method is not practical for separation of 5 gram samples! (125 prep injections for19 × 100mm column!)

20 Scale-up of Optimized HPLC Method – Theoretical AspectsWTRW2 = W02 + Wth Eq. 3= 16 N-1 t02 ( k) t02 k2 w ws-1As we mentioned before, in analytical scale-up experiments, the sample is hundred-times concentrated than the analytical one, and the column is often under overloading conditions. In the ideal case of mass overloading and partition mechanism, the bandwidth W of the overloading peaks can be expressed by this equation:In other words, the maximum loading was determined by retention factor and plate number before overloading, and the maximum fronting distance after overloading due to excessive sample weight. When the front of this desired peak first reached the base of this impurity peak, we call it touching-band separation. This equation allows the prediction of maximum loading for touching-band separation under PrepLC conditions.Just a reminder here: Overloading in real life is complicated, may include both mass overloading and volume overloading, and other phenomena, under which Eq. 3 may not be applicable.(column effect)(sample-weight effect)Ws: column saturation capacity ( ≈ 0.4 surface area)Overloading in reality may include mass overloading,volume overloading,and others.

24 PrepLC Purification - UV-triggered Fraction CollectionXBridge C18 (50 × 250 mm)30-60% CH3CN/NH4OH, 25 min,100 ml/min, 240 nm, 6.0 ml, 160 mg/ml.There are two ways to trigger the fraction collection in PrepLC. One is UV based and the other is MS based. This is an example of UV-triggered fraction collection. We also analyzed each fractions of this peak by injecting each of them back into analytical HPLC.

25 PrepLC Purification - Fraction Analysis & RecoveryAs you can see from this figure, all fractions were pure except the first one had a minor shoulder. From this calibration curve, the recovery is 97% if combining all fractions and 91 % without first fraction. The molecular ions of all fractions were 472.6, thus confirmed the specificity of the fractions.97 % Recovery (91 % without first fraction)

26 PrepLC Purification - MS-triggered Fraction CollectionIn mass-triggered fraction collection, the fraction collection is triggered by desired molecular weight. In this case, the fraction collection was triggered by appearance of molecular ions of

27 PrepLC Purification - MS-triggered Fraction CollectionThis is the zoom-in part of the previous fraction collection region. As you can see, one advantage of the MS-triggered fraction collection is that it can collect the desired mass part only, while neglecting the co-eluted part in the same peak. The final recovery is only 80%, which is lower that UV-triggered fraction collection.Recovery 80 %

28 Anal. Scale UpSummaryGradient RP analytical run is the best starting point for developing PrepLC methodScreening, optimization, and scale-up are effective steps toward PrepLC method development┐The best analytical methods are not always the best PrepLC methods, and scale-up experiments are imperative to validate the performance and loading of the analytical method under PrepLC conditionsUV-triggered fraction collection has high recovery and lower purity than MS-triggered fraction collection.ScreeningOptimizationAnal. Scale-upPrep LCHere comes the summary.

38 Method Optimization Summary for 02154-137We run the gradient screening, and under most gradient conditions we did not any separation for the isomers. We start to see separations with narrow gradients. For example, we saw Rs of 1.8 for 20-40% gradient.We also tried isocratic and it seemed isocratic conditions was a better choice in term of resolution. As you can see from this table, with the decrease of MeOH percentage, the isomer resolution increased. But decrease MeOH further after 20% did not improve the resolution significantly, although you see significant increase of retention.

40 From Scale-up to Gilson Separation for 02154-137(HE-TMK)XBridge C18, 4.6 × 100mm20 % MeOH/0.1% NH4OH20 min, 1 ml/min, 254 nm, 100 mg/mlInj Vol: 8 μl, 12.5 μl, 25 μlAgilent HP 1100(HE-TMK)XBridge C18, 50 × 250mm20 % MeOH/0.1% NH4OH50 min, 100 ml/min, 254 nm,Inj Vol: 1.5 (1.6) ml,100 mg/ml)GilsonTM LS SystemHere we found the touching-band separation was 8 ul. The scale-up factor from 4.6 by 100 to 19 by 250 column is about This means we inject 1600 ul in the prep column. Here is the the Gilson separation by injecting 1500 ul, which could be still considered as touching-band separation.But we were not satisfied by the touching-band loading. Only 150 mg, and we had 10 gram to separate. Besides, retention is too long, and total separation would take 400 L mobile phase.So we are still working on the possibility of normal phase separation.Touching-band loading still lowRetention still too largeNP HPLC in progress!